1. Field of the Invention
The present invention relates to the identification and use of positive regulators of alginate production in Pseudomonas aeruginosa. One aspect of the invention provides compositions and methods for the early detection and diagnosis of the conversion to mucoidy of Pseudomonas aeruginosa. The present invention also provides a molecular mechanism for detecting the conversion from the nonmucoid to the mucoid state, including molecular probes for the early detection of this disease state.
2. Background Art
Cystic Fibrosis (CF) is the most common inheritable lethal disease among Caucasians. The leading cause of high morbidity and mortality in CF patients are the chronic respiratory infections caused by Pseudomonas aeruginosa. Pseudomonas aeruginosa is an aerobic, motile, gram-negative bacterium with a simple metabolic demand that allows it to thrive in diverse environments. P. aeruginosa normally inhabits soil, water, and vegetation. Although it seldom causes disease in healthy people, P. aeruginosa is an opportunistic pathogen associated with fatal pneumonia in patients with CF, as well as patients with compromised immune systems and chronic infections such as non-cystic fibrosis bronchiectasis and urinary tract infections.
In CF patients, the initially colonizing P. aeruginosa strains are nonmucoid but in the CF lung, after a variable period, often one or two years, they inevitably convert into the mucoid form. Mucoid strains of P. aeruginosa grow as biofilms in the airways of CF patients (Yu, H., and N. E. Head, Front Biosci. 7:D442-57 (2002)). Biofilms refer to surface-attached bacterial communities encased in a glycocalyx matrix (Costerton, J. W., et al., Science 284:1318-22 (1999)). Mucoid P. aeruginosa biofilms are microcolonies embedded in a capsule composed of copious amounts of alginate, an exopolysaccharide (Govan, J. R., and V. Deretic, Microbiol. Rev. 60:539-74 (1996)) and are resistant to host defenses (Ramsey, D. M., and D. J. Wozniak, Mol. Microbiol. 56:309-22 (2005)).
The emergence of mucoid strains of P. aeruginosa in CF lungs signals the beginning of the chronic phase of infection and is associated with further disease deterioration and poor prognosis (Lyczak, J. B., et al., Clin. Microbiol. Rev. 15:194-222 (2002)). The chronic phase of infection due to P. aeruginosa is characterized by pulmonary exacerbations (fever, elevated white blood cell count, increased sputum production, and decreased pulmonary function) that require antimicrobial therapy (Miller, M. B., and Gilligan, P. H., J. Clin. Microbiol. 41:4009-4015 (2003)). CF exacerbations are typically interspersed with intervening periods of relative quiescence, with each phase lasting various lengths of time (Miller, M. B., and Gilligan, P. H., J. Clin. Microbiol. 41:4009-4015 (2003)). However, lung function continuously declines, the infecting strains become increasingly resistant, and inevitably, the patient succumbs to cardiopulmonary failure (Miller, M. B., and Gilligan, P. H., J. Clin. Microbiol. 41:4009-4015 (2003)).
There is a growing consensus that the lung pathology that occurs during chronic P. aeruginosa infection is due to a large extent to the immune response directed against pseudomonal biofilms (Miller, M. B., and Gilligan, P. H., J. Clin. Microbiol. 41:4009-4015 (2003)). High levels of cytokines and leukocyte-derived proteases can be detected in airway fluid from CF patients and are believed to be responsible for much of the lung damage that occurs in this patient population (Miller, M. B., and Gilligan, P. H., J. Clin. Microbiol. 41:4009-4015 (2003)). Alginate appears to protect P. aeruginosa from the consequences of this inflammatory response as it scavenges free radicals released by activated macrophages (Simpson, J. A., et al., Free Rad. Biol. Med. 6:347-353 (1989)). The alginate mucoid coating also leads to the inability of patients to clear the infection, even under aggressive antibiotic therapies, most probably because it provides a physical and chemical barrier to the bacterium (Govan and Deretic, Microbiol. Rev. 60:539-574 (1996)).
Early aggressive antibiotic treatment of the initial colonizing non-mucoid P. aeruginosa population might prevent or at least delay chronic pulmonary infection. However, questions still remain as to whether such treatment should be performed routinely or only during pulmonary exacerbation, and whether the regimen could potentially lead to the emergence of resistant strains (Ramsey and Wozniak, Mol. Microbiol. 56:309-322 (2005)). Since P. aeruginosa is inherently resistant to many antibiotics at concentrations that can be achieved in vivo, with the exception of ciprofloxacin, those to which it is sensitive need to be given intravenously (Wilson and Dowling, Thorax 53:213-219 (1998)). However, long-term, aggressive antibiotic treatment is not without side effects. Therefore, it would be more beneficial to place the emphasis on aggressive treatment strategies before the in vivo switch to mucoidy since once chronic infection is established, it is rarely possible to eradicate it even with intensive, antibiotic therapy. Thus, early detection of conversion to mucoidy in patients is desired to allow aggressive therapy, thereby preventing further disease deterioration.
Synthesis of alginate and its regulation has been the object of numerous studies (Govan, J. R., and V. Deretic, Microbiol. Rev. 60:539-74 (1996); Ramsey, D. M., and D. J. Wozniak, Mol. Microbiol. 56:309-22 (2005)). Alginate production is positively and negatively regulated in wild-type cells.
Three tightly linked genes algU, mucA, and mucB have been previously identified with a chromosomal region shown by genetic means to represent the site where mutations cause conversion to mucoidy (see U.S. Pat. Nos. 6,426,187, 6,083,691, 5,591,838, and 5,573,910, incorporated herein by reference in their entireties).
Positive regulation centers on the activation of the alginate biosynthetic operon (Govan, J. R., and V. Deretic, Microbiol. Rev. 60:539-74 (1996)). Positive regulators include the alternative stress-related sigma factor AlgU (Martin, D. W., et al., Proc. Natl. Acad. Sci. 90:8377-81 (1993)), also called AlgT (DeVries, C. A., and D. E. Ohman, J. Bacteriol. 176:6677-87 (1994)), and transcriptional activators AlgR and AlgB, which belong to a bacterial two component signaling system. The cognate kinase of AlgB is KinB (Ma, S., et al., J. Biol. Chem. 272:17952-60 (1997)) while AlgZ (Yu, H., et al., J. Bacteriol. 179:187-93 (1997)) may be the kinase that phosphorylates AlgR. However, unlike a typical two-component system, alginate overproduction is independent of phosphorylation of AlgR or AlgB (Ma, S., et al., J. Bacteriol. 180:956-68 (1998)).
Negative regulation of alginate has focused on the post-translational control of AlgU activity. In alginate regulation, the master regulator is AlgU and the signal transducer is MucA, a trans-inner membrane protein whose amino terminus interacts with AlgU to antagonize the activity of AlgU, and the carboxyl terminus with MucB, another negative regulator of alginate biosynthesis. The algUmucABC cluster is conserved among many Gram-negative bacteria. AlgU belongs to the family of extracytoplasmic function (ECF) sigma factors that regulate cellular functions in response to extreme stress stimuli. The action of ECF sigma factors is negatively controlled by MucA, MucB and MucC. This set of proteins forms a signal transduction system that senses and responds to envelope stress.
MucA is the anti-sigma factor that binds AlgU and antagonizes its transcriptional activator activity (Schurr, M. J., et al., J. Bacteriol. 178:4997-5004 (1996)). Consequently, inactivation of mucA in P. aeruginosa strain PAO1 results in the mucoid phenotype (Alg+) (Martin, D. W., et al., Proc. Natl. Acad. Sci. USA 90:8377-81 (1993); Mathee, K., et al., Microbiology 145:1349-57 (1999)). Clinical mucoid isolates of P. aeruginosa carry recessive mutations in mucA (Anthony, M., et al., J. Clin. Microbiol. 40:2772-8 (2002); Boucher, J. C., et al., Infect. Immun. 65:3838-46 (1997)). The transition from a non-mucoid to mucoid variant occurs in concurrence with the mucA22 allele after exposure to hydrogen peroxide, an oxidant in neutrophils (Mathee, K., et al., Microbiology 145:1349-57 (1999)).
MucB is located in the periplasm in association with the periplasmic portion of MucA (Mathee, K., et al., J. Bacteriol. 179:3711-20 (1997); Rowen, D. W., and V. Deretic, Mol. Microbiol. 36:314-27 (2000)). MucC is a mild negative regulator whose action is in synergy with MucA or MucB (Boucher, J. C., et al., Microbiology 143:3473-80 (1997)). MucD is a negative regulator whose dual functions include periplasmic serine protease and chaperone activities that are thought to help remove misfolded proteins of the cell envelope for quality control (Boucher, J. C., et al., J. Bacteriol 178:511-23 (1996); Yorgey, P., et al., Mol. Microbiol. 41:1063-76 (2001)).
Overproduction of alginate is an important virulence factor for bacterial biofilm formation in vivo. Alginate protects the bacterium from oxidative stress by scavenging the reactive oxygen species (Learn, D. B., et al., Infect. Immun. 55:1813-8 (1987); Simpson, J. A., et al., Free Radic. Biol. Med. 6:347-53 (1989)).
There is a significant and urgent need in hospitals and clinical laboratories for a rapid, sensitive and accurate diagnostic test for detection of potential conversion to mucoidy of P. aeruginosa prior to the detection of the emergence of a mucoid colony morphology on a growth plate in a laboratory.